Inhibiting inflammation with milk oligosaccharides

ABSTRACT

A method of inhibiting inflammation with milk oligosaccharides or glycoconjugates containing the oligosaccharides.

CROSS-REFERENCE TO RELATED APPLICATION PARAGRAPH

This application claims the benefit of U.S. Provisional Application No.61/223,145 filed on Jul. 6, 2009, the content of which is herebyincorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Various components in human milk, e.g., milk immunoglobulins,leukocytes, oligosaccharides, and glycoconjugates, protect infantsagainst infectious diseases. Human milk is thus considered a naturalefficacious “nutriceutical,” i.e., a model food that conveys immunologicbenefits.

Human milk has also been found to reduce the risk of developinginflammatory enteric diseases in infants. This anti-inflammationactivity has been attributed to the leukocytes, cytokines, andantioxidants in human milk. See Buescher, Adv Exp Med Biol. 501:207-22(2001).

SUMMARY OF THE INVENTION

The present invention is based on an unexpected discovery thatoligosaccharides in human milk inhibit inflammation.

Accordingly, one aspect of this invention features a method ofinhibiting inflammation by administering to a subject in need thereof aneffective amount of a composition containing one or more milk-derivedoligosacchairdes or one or more glycoconjugates containing theoligosaccharide(s). A milk-derived oligosaccharide contains a firstsugar unit (i.e., fucose, galactose, mannose, or sialic acid), which islocated at a non-reducing end of the oligosaccharide, and a second sugarunit (i.e., galactose, glucose, mannose, or N-acetylglucosamine), whichis directly linked to the first sugar unit. In one example, theoligosaccharide is a linear molecule having one non-reducing end and onereducing end. In another example, it is a branched molecule havingmultiple non-reducing ends and one reducing end. When theoligosaccharide has two non-reducing ends, the sugar unit at onenon-reducing end can be fucose and that at the other non-reducing endcan be fucose, galactose, or sialic acid, or alternatively, the sugarunit at one non-reducing end is sialic acid and that at the othernon-reducing end is galactose or sialic acid. The sugar unit at thereducing end can be a glucose or an N-acetylglucosamine.

The glycoconjugate(s) used in the method described above can include oneor more of the milk-derived oligosaccharide(s) also described aboveconjugated with a lipid, a peptide, a polypeptide, or a carbohydrate.

Another aspect of this invention features a method of inhibitinginflammation with oligosaccharides isolated from milk, which can bederived from a human, a bovid (e.g., a cow, a goat, or a sheep), oranother mammal (e.g. a horse or a camel). The oligosaccharides can beprepared by first removing fat and protein from the milk before itsisolation via conventional methods. In one example, after removal of fatand protein, the milk is loaded onto a carbon column and theoligosaccharides adsorbed onto the column are eluted with an alcoholsolution (e.g., a 50% aqueous ethanol solution).

The method of this invention can be applied to a subject, e.g., a humanor a non-human mammal, who is suffering from or at risk for developingan inflammatory disease, such as a disease of the digestive tract.Examples include oesophagitis, gastroenteritis, colitis, cholangitis,appendicitis, inflammatory bowel diseases (i.e., ulcerative colitis,necrotizing enterocolitis, and Crohn's disease), or irritable bowelsyndrome.

Also within the scope of this invention is use of one or moremilk-derived oligosaccharides or one or more glycoconjugates containingthe oligosaccharide(s) for inhibiting inflammation and for themanufacture of a medicament for treating inflammatory diseases.

The details of one or more embodiments of the invention are set forth inthe description below. Other features or advantages of the presentinvention will be apparent from the following drawings and detaileddescription of an example, and also from the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings are first described.

FIG. 1 is a chart showing that human milk oligosaccharides inhibit TNF-αinduced IL-8 production by T84 enterocytes.

FIG. 2 is a chart showing that human milk oligosaccharides inhibit TNF-αinduced monocyte chemoattractant protein-1 production by humanintestinal mucosa.

FIG. 3 is a chart showing that flagelin, polyinosinic-polycytidilicdouble-stranded RNA, or IL-1β induces IL-8 production in organ cultureof immature human intestinal mucosa and human milk oligosaccharidesinhibit this induced IL-8 production.

DETAILED DESCRIPTION OF THE INVENTION

Disclosed herein is a method of inhibiting inflammation with one or moremilk-derived oligosaccharides or one or more glycoconjugates containingthe oligosaccharides.

A milk-derived oligosaccharide, i.e., having at least three sugar units,is either a naturally-occurring oligosaccharide found in milk, afragment of the naturally-occurring oligosaccharide, or a variantthereof that contains a modified (e.g., sulfated, acetylated, orphosphorylated) sugar unit as compared to its natural counterpart. Thisoligosaccharide includes a non-reducing end motif S₁S₂, in which S₁ isfucose, galactose, mannose, or sialic acid (N-acetyl or N-glycolyl) andS₂ is galactose, glucose, mannose, or N-acetylglucosamine. S₁ is linkedto S₂ via an α or β glycosidic bond. When S₁ is fucose, the glycosidicbond between S₁ and S₂ preferably is an α1,2, an α1,3, or an α1,4 bond.When it is sialic acid, the glycosidic bond preferably is an α2,3 or anα2,6 bond.

Milk-derived oligosaccharides and glycolconjugates containing sucholigosaccharides are well known in the art. See, e.g., U.S. PatentApplication 61/168,674 and WO2005/055944. The following tables listexemplary oligosaccharides that naturally occur in human milk:

TABLE 1 Fucosyl oligosaccharides 2′FL 2-Fucosyllactose Fucα1,2Galβ1,4GlcLNF-I Lacto-N- Fucα1,2Galβ1,3GlcNAcβ1,3Galβ1,4Glc fucopentaose I LNF-IILacto-N- fucopentaose II

3′FL 3-Fucosyllactose

LNF-III Lacto-N- fucopentaose III

LDFH-I Lacto-N- difucohexaose I

LDFT Lactodifucotetraose

TABLE 2 Nonfucosylated, nonsialylated oligosaccharides LNTLacto-N-tetraose Galβ1, 3GlcNAcβ1, 3Galβ1, 4Glc LNneoTLacto-N-neotetraose Galβ1, 4GlcNAcβ1, 3Galβ1, 4Glc

TABLE 3 Sialyl milk oligosaccharide structures 3′-SL 3′-NANAα2,3Galβ1,4Glc Sialyllactose 6′-SL 6′- NANAα2,6Galβ1,4GlcSialyllactose SLNT-c Sialyllacto-N- NANAα2,6Galβ1,4GlcNAcβ1,3Galβ1,4Glcneotetraose c MSLNH Mono- sialyllacto- N-hexaose

DSLNH-I Disialyllacto- N-hexaose I

MSLNnH- I Mono- sialyllacto-N- neohexaose I

SLNnH-II Mono- sialyllacto-N- neohexaose II

DSLNnH Disialyllacto- N-neohexaose

DSLNT Disialyllacto- N-tetraose

DSLNH- II Disialyllacto- N-hexaose II

SLNT-a Sialyllacto- NANAα2,3Galβ1,3GlcNAcβ1,3Galβ1,4Glc N-tetraose aDSLNH-I Disialyllacto- N-hexaose I

SLNT-b Sialyllacto- N-tetraose b

TABLE 4 Sialyl fucosyl oligosaccharides 3′-S-3FL3′-Sialyl-3-fucosyllactose

DSFLNH Disialomonofucosyllacto-N-neohexaose

MFMSLNO Monofucosylmonosialyllacto-N-octaose (sialyl Lea)

SLNFH-II Sialyllacto-N-fucohexaose II

DSLNFP-II Disialyllacto-N-fucopentaose II

MFDLNT Monofucosyldisialyllacto-N-tetraose

The milk-derived oligosaccharides described herein can be prepared byconventional methods, e.g., synthesized chemically, purified from milk,or produced in a microorganism. See WO2005/055944. Below is an exampleof isolating oligosaccharides from milk. Milk is first defatted bycentrifugation to produce skimmed milk. The skimmed milk is then mixedwith an organic solvent, such as acetone (e.g., 50% aqueous acetone) andethanol (e.g., 67% aqueous ethanol), to precipitate milk proteins. Uponcentrifugation, the supernatant is collected and subjected tochromatography. Oligosaccharide-containing fractions are collected andpooled. If necessary, the oligosaccharides thus prepared can beconcentrated by conventional methods, e.g., dialysis or freeze-drying.

Milk oligosaccharides can also be isolated from skimmed milk by passingthe skimmed milk through a 30,000 MWCO ultrafiltration membrane,collecting the diffusate, passing the diffusate through a 500 MWCOultrafilter, and collecting the retentate, which contains milkoligosaccharides.

The glycoconjugates described herein, containing one or moremilk-derived oligosaccharides, can be chemically synthesized byconjugating the oligosaccharide(s) to a backbone molecule (e.g., acarbohydrate, a lipid, a nucleic acid, or a peptide) directly or via alinker. As used herein, “glycoconjugate” refers to a complex containinga sugar moiety associated with a backbone moiety. The sugar and thebackbone moieties can be associated via a covalent or noncovalent bond,or via other forms of association, such as entrapment (e.g., of onemoiety on or within the other, or of either or both entities on orwithin a third moiety). The glycoconjugate described herein can containone type of milk-derived oligosaccharide (i.e., one or more copies of amilk-derived oligosaccharide attached to one backbone molecule).Alternatively, the glycoconjugate contains multiple types ofmilk-derived oligosaccharides. In one example, the milk-derivedoligosaccharide (e.g., lacto-N-fucopentaose I, 2-fucosyllactose,lacto-N-difucohexaose I, lactodifucotetraose, or an acetylated variantthereof) is covalently linked via its reducing end sugar unit to alipid, a protein, a nucleic acid, or a polysaccharide. Preferably, thereducing end sugar unit is N-acetylglucosamine.

Peptide backbones suitable for making the glycoconjugate described aboveinclude those having multiple glycosylation sites (e.g., asparagine,lysine, serine, or threonine residue) and low allergenic potential.Examples include, but are not limited to, amylase, bile salt-stimulatedlipase, casein, folate-binding protein, globulin, gluten, haptocorrin,lactalbumin, lactoferrin, lactoperoxidase, lipoprotein lipase, lysozyme,mucin, ovalbumin, and serum albumin. Typically, a milk-derivedoligosaccharide can be covalently attached to a serine or threonineresidue via an O-linkage or attached to an asparagine residue via anN-linkage. To form these linkages, the sugar unit at the reducing end ofthe oligosaccharide is preferably an acetylated sugar unit, e.g.,N-acetylgalactosamine, N-acetylglucosamine, and N-acetylmannosamine. Anoligosaccharide can be attached to a peptide (e.g., a protein) usingstandard methods. See, e.g., McBroom et al., Complex Carbohydrates, PartB, 28:212-219, 1972; Yariv et al., Biochem J., 85:383-388, 1962;Rosenfeld et al., Carbohydr. Res., 46:155-158, 1976; and Pazur, Adv.Carbohydr. Chem, Biochem., 39:405-447, 1981.

In one example, a milk-derived oligosaccharide is linked to a backbonemolecule via a linker. Exemplary linkers are described in WO2005/055944.The oligosaccharide can be bonded to a linker by an enzymatic reaction,e.g., a glycosyltransferase reaction. A number of glycosyltransferases,including fucosyltransferases, galactosyltransferases,glucosyltransferases, mannosyltransferases, galactosaminyltransferases,sialyltransferases and N-acetylglucosaminyltransferases, can be used tomake the glycoconjugate described herein. More details about theseglycosyltransferases can be found in U.S. Pat. Nos.: 6,291,219;6,270,987; 6,238,894; 6,204,431; 6,143,868; 6,087,143; 6,054,309;6,027,928; 6,025,174; 6,025,173; 5,955,282; 5,945,322; 5,922,540;5,892,070; 5,876,714; 5,874,261; 5,871,983; 5,861,293; 5,859,334;5,858,752; 5,856,159; and 5,545,553.

Alternatively, the glycoconjugates described herein can be purified frommilk by conventional methods e.g., by passing through ultrafiltrationmembranes, by precipitation in non-polar solvents, or through partitionbetween immiscible solvents.

One or more of the above-described milk oligosaccharides orglycoconjugates can be mixed with a pharmaceutically acceptable carrierto form a pharmaceutical composition. The carrier in the pharmaceuticalcomposition must be “acceptable” in the sense of being compatible withthe active ingredient of the formulation (and preferably, capable ofstabilizing it) and not deleterious to the subject to be treated. Forexample, solubilizing agents such as cyclodextrins, which form moresoluble complexes with the oligosaccharides/glycoconjugates, or moresolubilizing agents, can be utilized as pharmaceutical carriers fordelivery of the oligosccharides/glyconjugates. Examples of othercarriers include colloidal silicon dioxide, magnesium stearate, sodiumlauryl sulfate, and D & C Yellow #10.

Alternatively, the oligoscchairdes/glycoconjugates can also beformulated as food produces or food supplements following methods wellknown in the food industry. In one example, they are components ofinfant formulas.

The oligosaccharides and glycoconjugates are effective in inhibitinginflammation and treating inflammation-associated diseases (i.e.,inflammatory diseases). Inflammation is reaction of living tissue (e.g.,heat, redness, swelling, or pain) in response to injury or infection.Exemplary inflammation-associated diseases, characterized by a local orsystemic, acute or chronic inflammation, include inflammatoryretinopathy (e.g., diabetic retinopathy), dermatoses (e.g., dermatitis,eczema, atopic dermatitis, allergic contact dermatitis, urticaria,necrotizing vasculitis, cutaneous vasculitis, hypersensitivityvasculitis, eosinophilic myositis, polymyositis, dermatomyositis, andeosinophilic fasciitis), hypersensitivity lung diseases (e.g.,hypersensitivity pneumonitis, eosinophilic pneumonia, delayed-typehypersensitivity, interstitial lung disease or ILD, idiopathic pulmonaryfibrosis, and ILD associated with rheumatoid arthritis), asthma, andallergic rhinitis. In addition to treating the above-listed inflammatorydiseases, the method of this invention is particularly effective intreating inflammatory disease of the digestive tract, includingoesophatigis (i.e., inflammation of the oesophagus, such as oesophagealulcer), gastroenteritis (i.e., inflammation of the mucous membranes ofthe stomach and intestine, such as gastritis, duodenal ulcer, ileitis,or enterocolitis), colitis (i.e., inflammation of the colon, such asdiverticulitis), cholangitis (i.e., inflammation of the bile duct), andappendicitis (i.e., inflammation of the appendix). Inflammatory diseaseof the digestive tract also includes inflammatory bowel diseases (e.g.,Crohn's disease and ulcerative colitis) and irritable bowel syndrome.

The term “treating” as used herein refers to the application oradministration of a composition including one or more active agents to asubject, who has an inflammatory disease, a symptom of the inflammatorydisease, or a predisposition toward the inflammatory disease, with thepurpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate,improve, or affect the disease, the symptoms of the disease, or thepredisposition toward the disease.

To practice the method of this invention, an effective amount of theabove-described pharmaceutical composition can be administered to asubject (e.g., a human infant or elderly) orally, parenterally, byinhalation spray, topically, rectally, nasally, buccally, vaginally orvia an implanted reservoir. The term “parenteral” as used hereinincludes subcutaneous, intracutaneous, intravenous, intramuscular,intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal,intralesional, and intracranial injection or infusion techniques. “Aneffective amount” as used herein refers to the amount of each activeagent required to confer therapeutic effect on the subject, either aloneor in combination with one or more other active agents. Effectiveamounts vary, as recognized by those skilled in the art, depending onroute of administration, excipient usage, and co-usage with other activeagents.

A sterile injectable composition, e.g., a sterile injectable aqueous oroleaginous suspension, can be formulated according to techniques knownin the art using suitable dispersing or wetting agents (such as Tween80) and suspending agents. The sterile injectable preparation can alsobe a sterile injectable solution or suspension in a non-toxicparenterally acceptable diluent or solvent, for example, as a solutionin 1,3-butanediol. Among the acceptable vehicles and solvents that canbe employed are mannitol, water, Ringer's solution and isotonic sodiumchloride solution. In addition, sterile, fixed oils are conventionallyemployed as a solvent or suspending medium (e.g., synthetic mono- ordiglycerides). Fatty acids, such as oleic acid and its glyceridederivatives are useful in the preparation of injectables, as are naturalpharmaceutically-acceptable oils, such as olive oil or castor oil,especially in their polyoxyethylated versions. These oil solutions orsuspensions can also contain a long-chain alcohol diluent or dispersant,or carboxymethyl cellulose or similar dispersing agents. Other commonlyused surfactants such as Tweens or Spans or other similar emulsifyingagents or bioavailability enhancers which are commonly used in themanufacture of pharmaceutically acceptable solid, liquid, or otherdosage forms can also be used for the purposes of formulation.

A composition for oral administration can be any orally acceptabledosage form including, but not limited to, capsules, tablets, emulsionsand aqueous suspensions, dispersions and solutions. In the case oftablets for oral use, carriers which are commonly used include lactoseand corn starch. Lubricating agents, such as magnesium stearate, arealso typically added. For oral administration in a capsule form, usefuldiluents include lactose and dried corn starch. When aqueous suspensionsor emulsions are administered orally, the active ingredient can besuspended or dissolved in an oily phase combined with emulsifying orsuspending agents. If desired, certain sweetening, flavoring, orcoloring agents can be added. A nasal aerosol or inhalation compositioncan be prepared according to techniques well known in the art ofpharmaceutical formulation.

Suitable in vitro and in vivo assays can be used to preliminarilyevaluate the anti-inflammation activity of a particular milkoligosaccharide or a combination of various milk oligosaccharides. Forexample, the oligosaccharide(s) can be tested in vitro for its abilityof inhibiting secretion of pro-inflammatory cytokines (e.g., IL-1, IL-6,TNF-alpha GM-CSF, IL-8, and IL-12). The anti-inflammation activity canfurther be confirmed in an animal model (e.g., a mouse model). Based onthe results, an appropriate dosage range and administration route canalso be determined.

Without further elaboration, it is believed that one skilled in the artcan, based on the above description, utilize the present invention toits fullest extent. The following specific example is therefore to beconstrued as merely illustrative, and not limitative of the remainder ofthe disclosure in any way whatsoever. All publications cited herein areincorporated by reference.

Use of Human Milk Oligosaccharides for Inhibiting IntestinalInflammation Preparation of Human Milk Oligosaccharides

An oligosaccharide fraction was isolated from human milk following themethod described in Chaturvedi et al., Anal. Biochem. 251(1):89-97,1997. Briefly, pooled human milk was first defatted and then ethanol wasadded to precipitate proteins. The resultant solution was loaded onto acarbon column, which adsorbs oligosaccharides. The column was washedwith 5% ethanol and the adsorbed oligosaccharides were eluted with 60%ethanol to produce a fraction containing human milk oligosaccharides(“HMOS”).

HMOS Inhibit IL-8 Secretion in TNF-treated T84 Cells

T84 cells, used routinely for studying neonatal epithelial inflammation,were cultured in 24-well Falcon organ culture dishes at 37° C. with 95%O₂ and 5% CO₂ in DMEM/F12 medium supplemented with FBS (5%), Hepesbuffer, NaOH, penicillin and streptomycin. These cells were treated with(i) saline as a negative control, (ii) TNF-α (10 ng/mL) as a positivecontrol, (iii) HMOS (5 g/L), and (iv) TNF-α (10 ng/mL) and HMOS (5 g/L).After 16 hours, the concentration of IL-8 in each culture supernatantwas measured by ELISA. The results thus obtained were standardized tothe cell numbers (i.e., divided by the total cell protein contents ofthe corresponding cell cutures).

As shown in FIG. 1, the TNF-induced IL-8 production was significantlyreduced in HMOS-treated T84 cells, indicating tht HMOS exhibitedanti-inflammatory activity.

HMOS Inhibit Monocyte Chemoattractant Protein-1 (MCP-1) Secretion inHuman Intestinal Mucosa

Human small intestine mucosa samples from 14 wk abortuses were incubatedin 24-well Falcon organ culture plates with CMRL 1066 mediumsupplemented with FBS (5%), glucose (5 g/L), tricine buffer (20 mM, pH7.4), hydrocortisone hemisuccinate (0.5 μL), β-retinyl acetate (1 mg/L),penicillin and streptomycin in 5% CO₂ at 37° C. The mucosa samples weretreated with (i) saline as a negative control, (ii) TNF-α (10 ng/mL) asa positive control, (iii) HMOS (5 g/L), and (iv) TNF-α (10 ng/mL) andHMOS (5 g/L). After 16 hours, the concentration of MCP-1, apro-inflammatory chemokine, was measured in each culture supernatant byELISA. The results thus obtained were standized to cell numbers asdescribed above.

The data obtained from this study, shown indicate that in the presenceof TNF-α, human intestinal mucosa secreted a high level of MCP-1, ameasure of inflammation and this TNF-αinduced MCP-1 production wasattenuated by HMOS. See FIG. 2.

Inhibition of IL-8 Secretion in Organ Culture of Immature HumanIntestinal Mucosa

Human small intestine samples from 22 wk abortuses were incubated in24-well plates with the modified CMRL media described above in 5% CO₂ at37° C. The samples were treated with IL-1β (10 ng/mL), flagellin (1mg/mL), polyinosinic-polycytidilic double stranded RNA (PIC; 10 ng/mL),or PBS (as a netative control) in the absence or presence of 5 mg/mLHMOS for 18 h. Levels of IL-8 secretion in the culture supernatants weremeasured using a ELISA kit (R & D Systems) in duplicate, with detectionat 450 nm on a versa max plate reader (Molecular Devices, Calif., USA).Each OD₄₅₀ value was normalized to the total protein amount of thecorresponding organ culture.

Flagelin, polyinosinic-polycytidilic double stranded RNA, and IL-1β allinduced a pro-inflammatory response, as evidenced by secretion of IL-8.See FIG. 3. This pro-inflammatory response was significantly attenuatedby HMOS.

Take together, the results shown above indicate that milkoligosaccharides are effective in inhibiting inflammation.

Other Embodiments

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features.

From the above description, one skilled in the art can easily ascertainthe essential characteristics of the present invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions. Thus, other embodiments are also within the claims.

1. A method of inhibiting inflammation, comprising administering to asubject in need thereof an effective amount of a composition containingat least a milk-derived oligosaccharide or a glycoconjugate containingthe oligosaccharide, the oligosaccharide including a first sugar unitand a second sugar unit, wherein the first sugar unit, located at afirst non-reducing end, is fucose, galactose, mannose, or sialic acidand the second sugar unit, linked to the first sugar unit, is galactose,glucose, mannose, or N-acetylglucosamine. 2.-31. (canceled)